褪黑素對小鼠卵母細(xì)胞成熟及早期胚胎發(fā)育的影響.pdf

1、Melatonin (MT) is a kind of indoles hormones，secreted from the pineal gland of brains in human and mammals mainly，and other organs and cells also secrete less. The production of MT is greater affected by illumination，and

2、 presents the circadian rhythm: commonly secrete more at night，less during the day. As effective antioxidants in vivo，MT has the character of highly lipophilic and hydrophilic，and can pass through the cytomembrane and a

3、variety of membrane structures into organelles to play the role of antioxidation. The main functions contain direct scavenging free radicals; improving the activity of antioxidant enzymes (including superoxide dismutase

4、(SOD); glutathione reductase (GSH-Px)) and decreasing the production of free radicals.At present there are many researches which study about the effect of MT as an antioxidant and antiapoptosis factor on the in vitro mat

5、uration of oocytes and early embryonic development in mice，pigs，sheep，chickens and other animals. However，the further studies of its molecular mechanism and receptor signaling pathways are reported less. In mice as test

6、object，this study aim to investigate the effect of oxidant hydrogen peroxide (H2O2) on mouse oocytes during IVM and the protection of MT against the oxidative damage; the influence of MT on embryo development after parth

7、enogenetic activation and early embryonic development in vitro. It will provide the theoretical and experimental basis for optimizing the system of embryo culture in vitro. The main contents can be stated as follows:1. T

8、he effect of melatonin on mouse oocytes threatened by H2O2 during IVM and parthenogenetic embryo development.The influence of H2O2 on the meiotic resumption and progression of mouse oocytes were assessed and the dates we

9、re summarized: The proportion of oocytes meiotic resumption (GVBD to MII) decreased with the addition of H2O2:the group of 100μM was no significant difference in comparison with the control group; The oxidative damage wa

10、s more and more obvious when more than 200μM，the discharge rate of PB1 was decreased significantly (P <0.05) when the concentration was increased to 250μM. There was no survival in 300μM .Screened the maximum concentrati

11、on for oxidative stress was 250μM.2. The protective effect of melatonin against the inhibitory effect of exogenous H2O2 on oocyte maturation.Different concentrations of MT (0,0.1,1,10ng/ml) were adding to the maturation

12、medium which containing 0μM，250μM of H2O2，After incubating for 12h，the PB1 discharged number of oocytes were analyzed. We found that when oocytes were cultured in the culture medium of the coexistence of MT and H2O2 (250

13、μM)，MT dose-dependently reversed the inhibition of H2O2 on oocytes maturation in vitro. It can be seen compared to the second group，the third，the four groups of maturation rate was significantly increased (42.0％ vs. 24.5

14、%; 47.4% vs. 24.5%，P <0.05)，the concentration of MT reached 10ng/mL，the reversal effect is the most significant (61.1%vs. 24.5%，P<0.05).3. The effect of MT on parthenogenetic development of mouse oocytes.In this experime

15、nt，different concentrations of MT (0，10-9，10-7，10-5，10-3M) were used to incubate parthenogenetic development of mouse oocytes. The results were shown that there was no significant difference in the groups of 10-9-10-3M t

16、han the control group during two-cell phase (88.2%，88.8%，91.4%，91.8%，88.8% vs. 88.1%，p>0.05)，the rate of four-eight-cell was improved when added 10-9M of MT (30.5%vs. 23.4%，p<0.05) and had emerged a significant differenc

17、e. There were very significant difference among the rates of 10-7-10-3M than control group (58.8%，73.8%，43.6% vs. 23.4%，p <0.05)，Blastocyst rates were increased in the higher concentrations 10-9M-10-5M (24.6%，46.6%，49.2%

18、 vs. 8.0%，p <0.05)，but it decreased during 10-3M of MT. In conclusion，the best concentration of MT for development of mouse oocytes after parthenogenetic activation was 10-5M.4. The effects of melatonin on mouse one-cell

19、 embryos development.Through experiment，we found that MT could overcome embryo developmental block，and significantly promote the embryonic development. When treated with 10-7，10-5M of MT，the blocking rates of two-cell we

20、re significantly increased (44.20%，41.60% vs. 26.6%，p <0.05)，but further development to the blastocyst was not increased under 10-5M. MT(10-9M,10-7M) increased development rates from four-cell to blastocyst (four-cell st

21、age，25.8%，25.3%vs.15.9%，p<0.05;eight-cell stage，23.4%，24.4% vs.12.1%，p <0.05),but not that of two-cell stage embryos. Moreover，a promoting effect in all stages from two-cell to blastocyst was observed at 10-7M (two-cell

22、stage，44.20%vs. 26.6%;four-cell stage 25.3%vs. 15.9%;eight-cell stage，24.4%vs. 12.1%;blastocyst，22.6%vs. 7.9%).5. The Hoechst 33342 staining of blastocyst.Blastocysts which were cultured at different concentrations of MT

23、 were stained by Hoechst 33342，the total cell numbers of blastocyst were observed. The higher concentration of MT could increase the blastocyst rate and total cell number of blastocyst. 10-7M of MT was the suitable conce

24、ntration for early embryo development，and were significantly increased the total cell number of blastocyst (42.7 vs. 32.4，p<0.05)，the other concentrations (10-9M; 10-5M; 10-3M) were not significantly than control group (

25、33.1;37.4;35.9 vs. 32.4).In conclusion，mouse oocytes were suffered oxidative damage by H2O2 which the highest concentration was 250μM. MT dose-dependently reversed the inhibition of H2O2 during IVM of oocytes. 10-7-10-5M